, Volume 230, Issue 4, pp 819–825 | Cite as

Identification of a plant-specific Zn2+-sensitive ribonuclease activity

  • Denghui Xing
  • Shuisong Ni
  • Michael A. Kennedy
  • Qingshun Quinn LiEmail author
Original Article


Ribonucleases (RNases) play a variety of cellular and biological roles in all three domains of life. In an attempt to perform RNA immuno-precipitation assays of Arabidopsis proteins, we found an EDTA-dependent RNase activity from Arabidopsis suspension tissue cultures. Further investigations proved that the EDTA-dependent RNase activity was plant specific. Characterization of the RNase activity indicated that it was insensitive to low pH and high concentration of NaCl. In the process of isolating the activity with cation exchange chromatography, we found that the EDTA dependency of the activity was lost. This led us to speculate that some metal ions, which inhibited the RNase activity, may be removed during cation exchange chromatography so that the nuclease activity was released. The EDTA dependency of the activity could be due to the ability of the EDTA chelating those metal ions, mimicking the effect of the cation exchange chromatography. Indeed, Zn2+ strongly inhibited the activity, and the inhibition could be released by EDTA based on both in-solution and in-gel assays. In-gel assays identified two RNase activity bands. Mass spectrometry assays of those activity bands revealed more than 20 proteins. However, none of them has an apparent known nuclease domain, suggesting that one or more of those proteins might possess a currently uncharacterized nuclease domain. Our results may shed light on RNA metabolism in plants by introducing a novel plant-specific RNase activity.


Ribonuclease Plant-specific RNase RNA metabolism EDTA dependency Arabidopsis thaliana 



We highly appreciate constructive suggestions of anonymous reviewers in terms of potential identity of the RNase activity. This work was supported in part by a grant from US National Institute of Health (1R15GM07719201A1 to QQL), and by a grant from Ohio Plant Biotech Consortium (to QQL and DX).

Supplementary material

425_2009_986_MOESM1_ESM.pdf (359 kb)
Supplementary material (PDF 359 kb)


  1. Addepalli B, Hunt AG (2007) A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor. Nucleic Acids Res 35:4453–4463PubMedCrossRefGoogle Scholar
  2. Addepalli B, Hunt AG (2008a) Redox and heavy metal effects on the biochemical activities of an Arabidopsis polyadenylation factor subunit. Arch Biochem Biophys 473:88–95PubMedCrossRefGoogle Scholar
  3. Addepalli B, Hunt AG (2008b) Ribonuclease activity is a common property of Arabidopsis CCCH-containing zinc-finger proteins. FEBS Lett 582:2577–2582PubMedCrossRefGoogle Scholar
  4. Backlund M, Paukku K, Daviet L, De Boer RA, Valo E, Hautaniemi S, Kalkkinen N, Ehsan A, Kontula KK, Lehtonen JY (2009) Posttranscriptional regulation of angiotensin II type 1 receptor expression by glyceraldehyde 3-phosphate dehydrogenase. Nucleic Acids Res 37:2346–2358PubMedCrossRefGoogle Scholar
  5. Bariola PA, Howard CJ, Taylor CB, Verburg MT, Jaglan VD, Green PJ (1994) The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J 6:673–685PubMedCrossRefGoogle Scholar
  6. Bariola PA, MacIntosh GC, Green PJ (1999) Regulation of S-like ribonuclease levels in Arabidopsis. Antisense inhibition of RNS1 or RNS2 elevates anthocyanin accumulation. Plant Physiol 119:331–342PubMedCrossRefGoogle Scholar
  7. Bonafé N, Gilmore-Hebert M, Folk NL, Azodi M, Zhou Y, Chambers SK (2005) Glyceraldehyde-3-phosphate dehydrogenase binds to the AU-Rich 3′ untranslated region of colony-stimulating factor-1 (CSF-1) messenger RNA in human ovarian cancer cells: possible role in CSF-1 posttranscriptional regulation and tumor phenotype. Cancer Res 65:3762–3771PubMedCrossRefGoogle Scholar
  8. Gerber AP, Luschnig S, Krasnow MA, Brown PO, Herschlag D (2006) Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc Natl Acad Sci USA 103:4487–4492PubMedCrossRefGoogle Scholar
  9. Gilbert C, Kristjuhan A, Winkler GS, Svejstrup JQ (2004) Elongator interactions with nascent mRNA revealed by RNA immunoprecipitation. Mol Cell 14:457–464PubMedCrossRefGoogle Scholar
  10. Green PJ (1994) The ribonucleases of higher plants. Annu Rev Plant Physiol Plant Mol Biol 45:421–445CrossRefGoogle Scholar
  11. Jarrous N, Reiner R (2007) Human RNase P: a tRNA-processing enzyme and transcription factor. Nucleic Acids Res 35:3519–3524PubMedCrossRefGoogle Scholar
  12. Ji X (2008) The mechanism of RNase III action: how dicer dices. Curr Top Microbiol Immunol 320:99–116PubMedCrossRefGoogle Scholar
  13. Keene JD, Komisarow JM, Friedersdorf MB (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc 1:302–307PubMedCrossRefGoogle Scholar
  14. Lers A, Sonego L, Green PJ, Burd S (2006) Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission. Plant Physiol 142:710–721PubMedCrossRefGoogle Scholar
  15. Liu X, Huang B, Lin J, Fei J, Chen Z, Pang Y, Sun X, Tang K (2006) A novel pathogenesis-related protein (SsPR10) from Solanum surattense with ribonucleolytic and antimicrobial activity is stress- and pathogen-inducible. J Plant Physiol 163:546–556PubMedCrossRefGoogle Scholar
  16. Mandel CR, Kaneko S, Zhang H, Gebauer D, Vethantham V, Manley JL, Tong L (2006) Polyadenylation factor CPSF-73 is the pre-mRNA 3′-end-processing endonuclease. Nature 444:953–956PubMedCrossRefGoogle Scholar
  17. Marchetti S, Zaina G, Chiaba C, Pappalardo C, Pitotti A (2001) Isolation and characterization of an endonuclease synthesized by barley (Hordeum vulgare L.) uninucleate microspores. Planta 213:199–206PubMedCrossRefGoogle Scholar
  18. Meyer S, Temme C, Wahle E (2004) Messenger RNA turnover in eukaryotes: pathways and enzymes. Crit Rev Biochem Mol Biol 39:197–216PubMedCrossRefGoogle Scholar
  19. Pantopoulos K (2004) Iron metabolism and the IRE/IRP regulatory system: an update. Ann N Y Acad Sci 1012:1–13PubMedCrossRefGoogle Scholar
  20. Ramachandran V, Chen X (2008a) Degradation of microRNAs by a family of exoribonucleases in Arabidopsis. Science 321:1490–1492PubMedCrossRefGoogle Scholar
  21. Ramachandran V, Chen X (2008b) Small RNA metabolism in Arabidopsis. Trends Plant Sci 13:368–374PubMedCrossRefGoogle Scholar
  22. Rodríguez-Pascual F, Redondo-Horcajo M, Magán-Marchal N, Lagares D, Martínez-Ruiz A, Kleinert H, Lamas S (2008) Glyceraldehyde-3-phosphate dehydrogenase regulates endothelin-1 expression by a novel, redox-sensitive mechanism involving mRNA stability. Mol Cell Biol 28:7139–7155PubMedCrossRefGoogle Scholar
  23. Rosenstierne MW, Vinther J, Mittler G, Larsen L, Mann M, Norrild B (2008) Conserved CPEs in the p53 3′ untranslated region influence mRNA stability and protein synthesis. Anticancer Res 28:2553–2559PubMedGoogle Scholar
  24. Ryan K, Calvo O, Manley JL (2004) Evidence that polyadenylation factor CPSF-73 is the mRNA 3′ processing endonuclease. RNA 10:565–573PubMedCrossRefGoogle Scholar
  25. Sikora D, Greco-Stewart VS, Miron P, Pelchat M (2009) The hepatitis delta virus RNA genome interacts with eEF1A1, p54(nrb), hnRNP-L, GAPDH and ASF/SF2. Virology 390:71–78PubMedCrossRefGoogle Scholar
  26. Sperling J, Azubel M, Sperling R (2008) Structure and function of the Pre-mRNA splicing machine. Structure 16:1605–1615PubMedCrossRefGoogle Scholar
  27. Tenenbaum SA, Carson CC, Lager PJ, Keene JD (2000) Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci USA 97:14085–14090PubMedCrossRefGoogle Scholar
  28. Tenenbaum SA, Lager PJ, Carson CC, Keene JD (2002) Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. Methods 26:191–198PubMedCrossRefGoogle Scholar
  29. Tong WH, Rouault TA (2007) Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis. Biometals 20:549–564PubMedCrossRefGoogle Scholar
  30. West S, Proudfoot NJ (2008) Human Pcf11 enhances degradation of RNA polymerase II-associated nascent RNA and transcriptional termination. Nucleic Acids Res 36:905–914PubMedCrossRefGoogle Scholar
  31. Xing D, Zhao H, Li QQ (2008) Arabidopsis CLP1-SIMILAR PROTEIN3, an ortholog of human polyadenylation factor CLP1, functions in gametophyte, embryo, and postembryonic development. Plant Physiol 148:2059–2069PubMedCrossRefGoogle Scholar
  32. Yan Q, Qi X, Jiang Z, Yang S, Han L (2008) Characterization of a pathogenesis-related class 10 protein (PR-10) from Astragalus mongholicus with ribonuclease activity. Plant Physiol Biochem 46:93–99PubMedCrossRefGoogle Scholar
  33. Yen Y, Green PJ (1991) Identification and properties of the major ribonucleases of Arabidopsis thaliana. Plant Physiol 97:1487–1493PubMedCrossRefGoogle Scholar
  34. Zemp I, Kutay U (2007) Nuclear export and cytoplasmic maturation of ribosomal subunits. FEBS Lett 581:2783–2793PubMedCrossRefGoogle Scholar
  35. Zhang J, Addepalli B, Yun KY, Hunt AG, Xu R, Rao S, Li QQ, Falcone DL (2008) A polyadenylation factor subunit implicated in regulating oxidative signaling in Arabidopsis thaliana. PLoS One 3:e2410PubMedCrossRefGoogle Scholar
  36. Zhou Y, Yi X, Stoffer JB, Bonafe N, Gilmore-Hebert M, McAlpine J, Chambers SK (2008) The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer. Mol Cancer Res 6:1375–1384PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Denghui Xing
    • 1
  • Shuisong Ni
    • 2
  • Michael A. Kennedy
    • 2
  • Qingshun Quinn Li
    • 1
    Email author
  1. 1.Department of BotanyMiami UniversityOxfordUSA
  2. 2.Department of Chemistry and BiochemistryMiami UniversityOxfordUSA

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